Elevated enhancer-oncogene contacts and higher oncogene expression levels by recurrent CTCF inactivating mutations in acute T cell leukemia.

Monoallelic inactivation of CCCTC-binding factor (CTCF) in human cancer drives altered methylated genomic states, altered CTCF occupancy at promoter and enhancer regions, and deregulated global gene expression. In patients with T cell acute lymphoblastic leukemia (T-ALL), we find that acquired monoallelic CTCF-inactivating events drive subtle and local genomic effects in nearly half of t(5; 14) (q35; q32.2) rearranged patients, especially when CTCF-binding sites are preserved in between the BCL11B enhancer and the TLX3 oncogene. These solitary intervening sites insulate TLX3 from the enhancer by inducing competitive looping to multiple binding sites near the TLX3 promoter. Reduced CTCF levels or deletion of the intervening CTCF site abrogates enhancer insulation by weakening competitive looping while favoring TLX3 promoter to BCL11B enhancer looping, which elevates oncogene expression levels and leukemia burden.

Improved Gene Fusion Detection in Childhood Cancer Diagnostics Using RNA Sequencing.

PURPOSE: Gene fusions play a significant role in cancer etiology, making their detection crucial for accurate diagnosis, prognosis, and determining therapeutic targets. Current diagnostic methods largely focus on either targeted or low-resolution genome-wide techniques, which may be unable to capture rare events or both fusion partners. We investigate if RNA sequencing can overcome current limitations with traditional diagnostic techniques to identify gene fusion events. METHODS: We first performed RNA sequencing on a validation cohort of 24 samples with a known gene fusion event, after which a prospective pan-pediatric cancer cohort (n = 244) was tested by RNA sequencing in parallel to existing diagnostic procedures. This cohort included hematologic malignancies, tumors of the CNS, solid tumors, and suspected neoplastic samples. All samples were processed in the routine diagnostic workflow and analyzed for gene fusions using standard-of-care methods and RNA sequencing. RESULTS: We identified a clinically relevant gene fusion in 83 of 244 cases in the prospective cohort. Sixty fusions were detected by both routine diagnostic techniques and RNA sequencing, and one fusion was detected only in routine diagnostics, but an additional 24 fusions were detected solely by RNA sequencing. RNA sequencing, therefore, increased the diagnostic yield by 38%-39%. In addition, RNA sequencing identified both gene partners involved in the gene fusion, in contrast to most routine techniques. For two patients, the newly identified fusion by RNA sequencing resulted in treatment with targeted agents. CONCLUSION: We show that RNA sequencing is sufficiently robust for gene fusion detection in routine diagnostics of childhood cancers and can make a difference in treatment decisions.

DNA Methylation Profiling Identifies Distinct Clusters in Angiosarcomas.

PURPOSE: DNA methylation profiling has previously uncovered biologically and clinically meaningful subgroups within many tumor types, but was not yet performed in angiosarcoma. Angiosarcoma is a rare sarcoma with very heterogeneous clinical presentations, which may be based on differences in biological background. In this exploratory study, DNA methylation profiling of 36 primary angiosarcoma samples from visceral, deep soft tissue, radiation-induced, and UV-induced localizations was performed. EXPERIMENTAL DESIGN: Primary angiosarcoma formalin-fixed paraffin-embedded samples from visceral, soft tissue, radiation-induced, and UV-induced origin were collected from a nationwide search for angiosarcoma in the Netherlands. DNA was extracted for methylation profiling with the Illumina Infinium MethylationEPIC array. Quality control assessment and unsupervised hierarchical clustering were performed. Copy-number profiles were generated and analyzed for chromosomal stability. Clinical data were obtained from the Netherlands Cancer Registry. RESULTS: DNA methylation profiling by unsupervised hierarchical clustering of 36 angiosarcoma samples (6 visceral, 5 soft tissue, 14 radiation-induced, 11 UV-induced) revealed two main clusters (A and B), which were divided into four subclusters. The clusters largely corresponded with clinical subtypes, showing enrichment of UV-induced cases in cluster A1 and radiation-induced cases in cluster A2. Visceral and soft tissue cases almost exclusively fell into cluster B. Cluster A showed significantly increased chromosomal instability and better overall survival (22 vs. 6 months, P = 0.046) compared with cluster B. CONCLUSIONS: In this novel methylation profiling study, we demonstrated for the first time four different angiosarcoma clusters. These clusters correlated with clinical subtype, overall survival, and chromosomal stability.

Kaposiform hemangioendothelioma and tufted angioma - (epi)genetic analysis including genome-wide methylation profiling.

Kaposiform hemangioendothelioma (KHE) is a locally aggressive vascular condition of childhood and is clinicopathologically related to tufted angioma (TA), a benign skin lesion. Due to their rarity molecular data are scarce. We investigated 7 KHE and 3 TA by comprehensive mutational analysis and genome-wide methylation profiling and compared the clustering, also with vascular malformations. Lesions were from 7 females and 3 males. The age range was 2 months to 9 years with a median of 10 months. KHEs arose in the soft tissue of the thigh (n = 2), retroperitoneum (n = 1), thoracal/abdominal (n = 1), supraclavicular (n = 1) and neck (n = 1). One patient presented with multiple lesions without further information. Two patients developed a Kasabach-Merritt phenomenon. TAs originated in the skin of the shoulder (n = 2) and nose/forehead (n = 1). Of the 5 KHEs and 2 TAs investigated by DNA sequencing, one TA showed a hot spot mutation in NRAS, and one KHE a mutation in RAD50. Unsupervised hierarchical clustering analysis indicated a common methylation pattern of KHEs and TAs, which separated from the homogeneous methylation pattern of vascular malformations. In conclusion, methylation profiling provides further evidence for KHEs and TAs potentially forming a spectrum of one entity. Using next generation sequencing, heterogeneous mutations were found in a subset of cases (2/7) without the presence of GNA14 mutations, previously reported in KHE and TA.